A diagnostic real-time PCR assay for the rapid identification of the tomato-potato psyllid, Bactericera cockerelli (Šulc, 1909) and development of a psyllid barcoding database

Abstract

The accurate and rapid identification of insect pests is an important step in the prevention and control of outbreaks in areas that are otherwise pest free. The potato-tomato psyllid Bactericera cockerelli (Š ulc, 1909) is the main vector of 'Candidatus Liberibacter solanacearum' on potato and tomato crops in North America and New Zealand; and is considered a threat for introduction in Europe and other pest-free regions. This study describes the design and validation of the first species-specific TaqMan probe-based real-time PCR assay, targeting the ITS2 gene region of B. cockerelli. The assay detected B. cockerelli genomic DNA from adults, immatures, and eggs, with 100% accuracy. This assay also detected DNA from cloned plasmids containing the ITS2 region of B. cockerelli with 100% accuracy. The assay showed 0% false positives when tested on genomic and cloned DNA from 73 other psyllid species collected from across Europe, New Zealand, Mexico and the USA. This included 8 other species in the Bactericera genus and the main vectors of 'Candidatus Liberibacter solanacearum' worldwide. The limit of detection for this assay at optimum conditions was 0.000001ng DNA (~200 copies) of ITS2 DNA which equates to around a 1:10000 dilution of DNA from one single adult specimen. This assay is the first real-time PCR based method for accurate, robust, sensitive and specific identification of B. cockerelli from all life stages. It can be used as a surveillance and monitoring tool to further study this important crop pest and to aid the prevention of outbreaks, or to prevent their spread after establishment in new areas.

Illuminating Insights into the Biodiversity of the Australian Psyllids (Hemiptera: Psylloidea) Collected Using Light Trapping

“…However, for the remaining 67 taxa, closely matching COI sequences were not available on GenBank or BOLD. This shows the lack of a comprehensive psyllid COI database present online and highlights the importance of generating sequences from both unidentified species and species with a confirmed taxonomical identification, as has recently been suggested elsewhere [55]. The need for a complete reference database is highlighted in our study, not only to better understand biodiversity, but also for biosecurity-two of the species we collected had their closest matches on GenBank with a major exotic pest, the Asian citrus psyllid Diaphorina citri (Table S1).…”

Molecular diagnostics of Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillaridae)

“…The specific diagnostic methods used or quantitative-PCR methods. These are commonly used in molecular diagnostics providing ever quicker turnaround times (Davis et al, 2011;Dhami et al, 2016;Sumner-Kalkun et al, 2020;Valenzuela et al, 2007).…”

“…If Sanger sequencing is infeasible, a range of additional molecular based diagnostic methods can be developed such as PCR‐RFLP (Polymerase Chain Reaction‐Restriction Fragment Length Polymorphism) or quantitative‐PCR methods. These are commonly used in molecular diagnostics providing ever quicker turnaround times (Davis et al, 2011; Dhami et al, 2016; Sumner‐Kalkun et al, 2020; Valenzuela et al, 2007). But these do require access to a dedicated molecular laboratory and specialist staff, which is not always possible.…”

Advances in insect pest and disease monitoring and forecasting in horticulture

“…The bottlenecks lie in getting sufficient amounts of DNA easily in field conditions and having to send the DNA samples to a laboratory for processing. Rapid identification tools based on extremely small amounts of DNA are available for laboratory use, as for example, for eggs, immatures and adults of a psyllid pest of potato (Sumner-Kalkun et al, 2020). DNA can be extracted from trapped individuals with Flinders Technology Associates FTA®cards, that is, chemically treated filter papers designed for the collection, preservation and shipment of biological samples for subsequent DNA and RNA analysis.…”